4-N-acylfortimicin B derivatives and the chemical conversion of fortimicin B to fortimicin A

ABSTRACT

This invention provides 4-N-acylfortimicin B derivatives of the structure ##STR1## wherein R is acyl, aminoacyl, N-monoloweralkylaminoacyl, N,N-diloweralkylaminoacyl, hydroxy-substituted aminoacyl, or substituted aminoacyl of the formula ##STR2## where R 1  is an acyl radical derived from an amino acid or a short peptide, and the pharmaceutically acceptable salts thereof. 
     The compounds are useful as intermediates for preparing 4-N-alkyl or substituted alkylfortimicin B derivatives. In addition to their utility as intermediates, some of the compounds of this invention are also useful as antimicrobial agents.

BACKGROUND OF THE INVENTION

This is a division of application Ser. No. 725,829 filed Sept. 23, 1976,now U.S. Pat. No. 4,091,032.

Antibiotic therapy plays a vial role in modern medicine. The advent ofantibiotic therapy in this century has, in part, been responsible forthe increased life expectancy, as well as lower instances of infant andchildbirth deaths. While there are numerous classes of antibioticsavailable, the semi-synthetic penicillins, the tetracyclines,erythromycins and cephalosporins are probably the most widely usedantibiotics.

Despite the availability of a variety of highly effectively antibiotics,the search for improved agents is a continuing one for a variety ofreasons. Many organisms become resistant to a particular antibiotic orclass of antibiotics and thus new drug entities must be continually madeavailable to treat infections involving strains of organisms which havebecome resistant to all other therapy. Apart from the problem ofresistance, this powerful class of drugs have a number of undesirableside effects and thus the search continues for agents which are lower intoxicity than presently available antibiotics yet are effectiveantimicrobial agents.

Another problem with current antibiotic therapy is that there arecertain organisms, such as the genus proteus of organisms, which arevery difficult to treat. Thus researchers are constantly seeking newentities which would be effective against various proteus strains.

Recently a new class of antibiotics has been identified and designatedas the fortimicins. To date, two fortimicin antibiotics are known,fortimicin A and fortimicin B. Both antibiotics are fermentationproducts and thus are difficult and expensive to manufacture.

Fortimicin A exhibits a wide range of in vitro activity againstgram-positive and gram-negative bacteria and also exhibits excellentactivity against strains of Staphylococcus aureus and Escherichia coliwhich is resistant to various known antibiotics such as kanamycin,gentamicin, tobramycin and the like, as well as exhibiting antibacterialactivity against bacteria of the genus Proteus. In vivo tests indicatethe ED₅₀ of fortimicin A against Escherichia coli Juhl KY 4286 in miceto be 6 mg./kg. (See U.S. Pat. No. 3,976,768).

Fortimicin B also exhibits in vitro antibacterial activity againstvarious gram-positive and gram-negative antibiotics, but is considerablyless active than fortimicin A. (See U.S. Pat. No. 3,931,400).

While fortimicin A is a promising lead in the class of fortimicinantibiotics, it has been found that the 4-N-alkylfortimicin Bderivatives are generally more stable, but just as effective asfortimicin A.

The present invention provides a novel series of intermediates which areuseful in preparing the 4-N-alkylfortimicin derivatives and alsoprovides a method of converting fortimicin B to fortimicin A.

SUMMARY OF THE INVENTION

This invention provides a novel series of 4-N-acylfortimicin Bderivatives which are useful as intermediates in the synthesis of4-N-alkylfortimicin B derivatives. In addition to their utility asintermediates, some of the compounds of this invention, as shown inTable II are also useful as antimicrobial agents.

This invention also provides a method for the chemical conversion of theless active fortimicin B to fortimicin A, as well as 4-N-acylfortimicinB derivatives.

Generally speaking the 4-N-alkylfortimicin B derivatives are prepared byreducing the acyl amide function of the particular 4-N-acylfortimicin Bderivative with for example lithium aluminum hydride or diborane whichare standard amide reduction procedures. The compounds of this inventionare used as intermediates in the synthesis of 4-N-substitutedalkylfortimicin B derivatives as well as 4-N-alkylfortimicin Bderivatives. Specifically, in addition to the 4-N-alkyl derivatives,they are useful in preparing 4-N-aminoalkyl or 4-N-hydroxyalkylderivatives of fortimicin B.

The present invention also provides for the chemical conversion offortimicin B (1) of the formula ##STR3## to fortimicin A (2) of theformula ##STR4## where R is ##STR5## and the preparation of fortimicin Aanalogs (4-N-acylfortimicin B derivatives) in which the 4-N-glycyl groupof the naturally occurring aminocyclitol antibiotic, fortimicin A (2),is replaced by acyl groups derived from carboxylic acids and amino acidsother than glycine where R is as defined above. In particular, theinvention is concerned with the preparation of 4-N-acylfortimicin Bderivatives in which the 4-N-acyl group is derived from an amino acid ora peptide, and their pharmaceutically acceptable salts.

DETAILED DESCRIPTION OF THE INVENTION

This invention is related to novel fortimicins and more particularly to4-N-acylfortimicin B derivatives, and to the chemical conversion offortimicin B to fortimicin A. The compounds of the present invention arerepresented by the formula ##STR6## wherein R is acyl, aminoacyl,N-monoloweralkylaminoacyl, N,N-diloweralkylaminoacyl,hydroxy-substituted aminoacyl, or substituted aminoacyl of the formula##STR7## where R¹ is an acyl radical derived from an amino acid or ashort peptide, and the pharmaceutically acceptable salts thereof.

These compounds are useful as intermediates for preparing 4-N-alkyl orsubstituted alkylfortimicin B derivatives. In addition to the utilitiesas intermediates, some of the compounds of this invention are alsouseful as antimicrobial agents.

The term "acyl" as used herein, refers to groups R represented by theformula ##STR8## wherein R² is loweralkyl, aminoloweralkyl,N-substituted-aminoloweralkyl and N,N-disubstituted-aminoloweralkylwherein the N-substituents of the N-substituted-aminoloweralkyl andN,N-disubstituted-aminoloweralkyl groups are comprised of alkyl groupssuch as methyl and ethyl. The term "lower alkyl" refers to both straightand branched chain C₁ -C₇ alkyl groups.

In addition, the term "acyl" as used herein, refers to groups Rrepresented by the formula ##STR9## wherein R¹ is an acyl radicalderived from an amino acid or a short peptide.

In addition, the acyl groups are derived from naturally occurring aminoacids or their enantiomers, which are not included among those definedabove, such as histidine, phenylalanine, tyrosine, or small peptidessuch as glycylglycine or other di- or tri-peptides.

As used here, the term "Cbz" refers to benzyloxycarbonyl.

The term "pharmaceutically acceptable salts", as used herein, refers tothe non-toxic acid addition salts which are generally prepared byreacting the compounds of this invention with a suitable organic orinorganic acid. Representative salts include the hydrochloride,hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate,laurate, borate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napsylate and the like.

The method illustrated below, which may be used for the preparation offortimicin A (2) from fortimicin B (1) and also for the preparation ofthe fortimicin A analogs (5) involves as the first step the preparationof 1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) by treatment offortimicin B (1) with a suitable acylating agent such asN-(benzyloxycarbonyloxy) succinimide (6),benzyloxycarbonyl-p-nitrophenol (8), respectively. ##STR10## in asolvent such as N,N-dimethylformamide, methanol-water, and the likeaccording to Scheme 1: ##STR11##

The second step of the process, the acylation of the C₄ -N-methylaminogroup of 1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) is accomplishedwith an activated carboxylic acid derivative such as carboxylic acidanhydride, a carboxylic acid chloride, an active carboxylic acid ester,or a carboxylic acid azide following the methodology commonly used inpeptide synthesis.

The active esters may be prepared from the carboxylic acid derivative##STR12## with 1-hydroxybenzotriazole, N-hydroxysuccinimide, orN-hydroxy-5-norbornene-2,3-dicarboximide [M. Fujino, S. Kobayashi, M.Obayashi, T. Fukuda, S. Shinagawa, and O. Nishimura, Chem. Pharm. Bull.Japan, 22, 1857 (1974)] respectively, as illustrated in Schemes A, B andC, below, wherein ##STR13## is acyl, N,N-diloweralkylaminoacyl, or anacyl group derived from an N-benzyloxycarbonyl protected amino acid or ashort peptide. ##STR14##

The reactions of the active esters with1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) are carried out in aninert solvent such as tetrahydrofuran, dioxane, chloroform,N,N-dimethyl-formamide and the like. In some cases, the addition of atertiary amine, such as triethylamine, proves beneficial.

In some of the couplings, the azide group is used to activate thecarboxyl terminal of the carboxylic acid to be coupled. The acyl azidesare made from the corresponding acyl hydrazides with HNO₂ (nitrousacid), and the excess acid is removed by a basic aqueous wash. Thereaction is illustrated below. ##STR15## where ##STR16## represents thesame groups as in the active ester preparation above. The couplingreactions of the acyl azides prepared above with1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) are carried out in aninert solvent such as ethyl acetate.

The coupling reactions of the above N-protected carboxyl activatedderivatives at the C₄ -N-methyl group of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) to form4-N-acyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B (4) is illustratedin Scheme 2 below: ##STR17## where Y represents activating groups suchas: ##STR18## and R⁴ is ##STR19## as defined above.

To those skilled in the art of peptide synthesis it is obvious that theintroduction of a short N-protected peptide chain in 3 to afford 4 maybe achieved in a stepwise manner by using suitably protectedintermediates as illustrated in Scheme 3 below: ##STR20## wherein R⁴ is##STR21## is as defined earlier and Y is an activating group as definedabove.

The stepwise synthesis proceeds via4-N-(N-tert-butyloxycarbonylglycyl)-1,2',6'-tri-N-benzyloxycarbonylfortimicinB (40) which under acidic conditions, such as trifluoroacetic acid inmethylene chloride, gives rise to4-N-glycyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B trifluoroacetatesalt (41). The latter (41) is first treated with triethylamine and thenallowed to react in the usual manner with ##STR22## to yield the4-N-acyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B (4) intermediatesin a stepwise procedure.

After completion of the acylation at the C₄ -N-methyl group of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) to form the protectedintermediates 4, it is necessary to remove the benzyloxycarbonylprotecting groups of 4 by hydrogenolysis of the latter (4) over apalladium on carbon catalyst to obtain the biologically activefortimicin A analogs (5). Fortimicin A (2) and the fortimicin A analogs(5) thus prepared are conveniently isolated as the hydrochloride saltswhen the hydrogenolyses are carried out in the presence of a slightexcess of hydrochloric acid. The hydrogenolyses of 4 to obtain 5 areformulated in Scheme 4 below: ##STR23## wherein R⁴ and R are as definedabove.

The compounds which may be prepared according to the method describedabove include the compounds represented by the formula ##STR24## where Ris as defined above. Examples of such compounds, which are not meant tolimit the scope of the invention, are the following:

(9) the tetrahydrochloride salt of fortimicin A, where R is ##STR25##(10) the tetrahydrochloride salt of4-N-(DL-2-hydroxy-4-aminobutyryl)fortimicin B where R is ##STR26## (11)the trihydrochloride salt of 4-N-acetylfortimicin B where R is ##STR27##(12) the tetrahydrochloride salt of 4-N-glyclglycylfortimicin B where Ris ##STR28## (13) the tetrahydrochloride salt of 4-N-sarcosylfortimicinB where R is ##STR29## (14) the tetrahydrochloride salt of4-N-L-phenylalanylglycylfortimicin B where R is ##STR30## (15) thetetrahydrochloride salt of 4-N-(N,N-dimethylglycyl) fortimicin B where Ris ##STR31## (16) the tetrahydrochloride salt of 4-N-β-alanylfortimicinB where R is ##STR32## (17) the tetrahydrochloride salt of4-N-D-alanylfortimicin B where R is ##STR33## (18) thetetrahydrochloride salt of 4-N-L-alanylfortimicin B where R is ##STR34##(19) the tetrahydrochloride salt of 4-N-L-alanylglycylfortimicin B whereR is ##STR35## (20) the tetrahydrochloride salt of4-N-L-leucylglycylfortimicin B where R is ##STR36## (21) thetetrahydrochloride salt of4-N-(DL-2-hydroxy-4-aminobutyryl)glycylfortimicin B where R is ##STR37##(22) the pentahydrochloride salt of 4-N-L-histidylfortimicin B where Ris ##STR38## (23) the tetrahydrochloride salt of4-N-glycylglycylglycylfortimicin B where R is ##STR39## (24) thetetrahydrochloride salt of4-N-(DL-2-hydroxy-3-aminopropionyl)glycylfortimicin B where R is##STR40## (25) the tetrahydrochloride salt of4-N-(DL-2-hydroxy-3-aminopropionyl)fortimicin B where R is

The following examples are provided to further illustrate the presentinvention and are not intended to limit or restrict the invention.

EXAMPLE 1 1,2',6'-Tri-N-benzyloxycarbonylfortimicin B (3)

To a stirred solution of 2.0 g. of fortimicin B (1), 30 ml. of water,and 60 ml. of methanol, cooled in an ice bath at 0°, was added 4.44 g.of N-(benzyloxycarbonyloxy)succinimide. Stirring was continued at 0° for3 hours and then at ambient temperature for 22 hours. The major portionof the methanol was evaporated under reduced pressure and the residuewas shaken with a mixture of chloroform and water. The chloroformsolution was washed with water and dried over anhydrous magnesiumsulfate. The chloroform was evaporated and the residue waschromatographed on silica gel. Elution with a solvent system composed ofchloroform-methanol-concentrated ammonium hydroxide (23.4:1.4:0.1 v/v)yielded 1.05 g. of 1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3):[α]_(D) ²⁵ + 16.5° (C 1.0, CH₃ OH); IR 1712, 1507 cm⁻¹ ; NMR (CDCl₃) δ1.03 (C₆ '--CH₃, J=6.0), 2.32 (NHCH₃), 3.41 (OCH₃).

Analysis Calcd. for: C₃₉ H₅₀ N₄ O₁₁ : C, 62.39; H, 6.71; N, 7.46. Found:C, 62.16; H, 6.76; N, 7.43.

EXAMPLE 2 Tetra-N-benzyloxycarbonylfortimicin A (26)

A. To a magnetically stirred solution of 1.00 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.357 g. ofN-benzyloxycarbonylglycine and 0.376 g. of 1-hydroxybenzotriazolemonohydrate in 2.8 ml. of tetrahydrofuran, cooled to 0° in an ice bath,was added a solution of 0.353 g. of N,N'-dicyclohexylcarbodiimide in 2.8ml. of tetrahydrofuran. An additional 2.8 ml. of tetrahydrofuran wasadded to rinse all the N,N-dicyclohexylcarbodiimide into the reactionvessel. Stirring was continued at 0° for 1 hour and then at ambienttemperature for 18 hours. The precipitated N,N'-dicyclohexylurea wasremoved by filtration. The tetrahydrofuran was evaporated from thefiltrate under reduced pressure leaving 1.79 g. of product. A sample(1.20 g.) was chromatographed on a column of silica gel, prepared andeluted with a solvent system consisting of benzene-methanol-95%ethanol-concentrated ammonium hydroxide (23.5:1.4:2.0:0.2 v/v).Fractions containing the desired product were combined and concentratedunder reduced pressure leaving 0.826 g. oftetra-N-benzyloxycarbonylfortimicin A: [α]_(D) ²³ + 52.9° (C 1.0, CH₃OH); IR 1710, 1635, 1500 cm⁻¹ ; NMR (CDCl₃) δ 1.16 (C₆ '--CH₃, J=6.5),2.82 (C₄ --NCH₃), 3.31 (OCH₃), 4.80 (H₁ ', J=3.0).

Analysis Calcd: for: C₄₉ H₅₉ N₅ O₁₄ : C, 62.48; H, 6.31; N, 7.43. Found:C, 62.52; H, 6.49; N, 7.23.

B. To a magnetically stirred solution of 4.02 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B in 40 ml. oftetrahydrofuran, cooled to 0° in an ice bath, was added 1.80 g. of theN-hydroxysuccinimide ester of N-benzyloxycarbonylglycine. Stirring wascontinued at 0° for 4 hours and then at room temperature for 23 hours.The resulting solution was shaken with a mixture of 300 ml. of CHCl₃ and400 ml. of 5% aqueous NaHCO₃ solution. The CHCl₃ solution was separatedand washed with 400 ml. of water. The aqueous solutions were washed inseries with three 200 ml. portions of CHCl₃. The CHCl₃ was evaporatedunder reduced pressure to yield 5.18 g. of a white glass. This productwas chromatographed on a column of 250 g. of silica gel (3.4 × 74 cm.).Elution was carried out with a solvent system composed ofbenzene-methanol-ethanol-ammonium hydroxide (23.5:1.60:1.80:0.20 v/v).The fractions containing the product were combined, and evaporation ofthe solvent left 4.58 g. of tetra-N-benzyloxycarbonylfortimicin A (26)identical with that prepared as described above.

EXAMPLE 3Tetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-4-aminobutyryl)fortimicin B(27)

To a magnetically stirred solution of 1.03 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.693 g. ofN-benzyloxycarbonyl-DL-1-hydroxy-4-aminobutyric acid, and 0.829 g. of1-hydroxybenzotriazole monohydrate in 5 ml. of tetrahydrofuran, cooledin an ice bath, was added a solution of 0.560 g. ofN,N'-dicyclohexylcarbodiimide in 2.5 ml. of tetrahydrofuran. Anadditional 2.5 ml. of tetrahydrofuran was added to rinse all of theN,N'-dicyclohexylcarbodiimide into the reaction vessel. Stirring wascontinued for 15 minutes in the ice bath and 0.8 ml. of triethylaminewas then added. Stirring was continued at 0° for 15 minutes and then atambient temperature for 21.5 hours. Insoluble N,N-dicyclohexylurea wasseparated by filtration and the tetrahydrofuran was removed from thefiltrate leaving 2.91 g. of a yellow glass. Chromatography was carriedout first on a silica gel column by eluting with a solvent systemcomposed of benzene-methanol-ethanol-concentrated ammonium hydroxide(23.5:0.7:2.7:0.2 v/v). Fractions enriched in the desired product werecombined and rechromatographed on silica gel using a solvent systemcomposed of benzene-methanol-ethanol (23.5:0.7:2.7 v/v). Fractionsenriched in the desired product were then chromatographed on SephadexLH-20 in methanol to yield 0.353 g. oftetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-4-aminobutyryl)fortimicin B(27): [α]_(D) ²⁴ + 42.4° (C 1.0, CH₃ OH); IR 1705, 1623, 1504 cm⁻¹ ; NMR(CDCl₃) δ 1.19 (C₆ '--CH₃), 2.9 (C₄ --NCH₃), 3.32 (OCH₃), 4.75 (H₁ ',J=3.0).

Analysis Calcd. for: C₅₁ H₆₃ N₅ O₁₅ : C, 62.12; H, 6.44; N, 7.10. Found:C, 62.07; H, 6.54; N, 7.07.

EXAMPLE 4 1,2',6'-Tri-N-benzyloxycarbonyl-4-N-acetylfortimicin B (28)

To a stirred solution of 3.22 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) in 225 ml. of methanol,cooled in an ice bath, was added 16 ml. of acetic anhydride over aperiod of 15 minutes. Stirring was continued at 0° for 2 hours and thenat room temperature for 2 hours. The methanol was evaporated underreduced pressure and residual acetic anhydride and acetic acid wereremoved by co-distillation with benzene and methanol to leave 3.63 g. of1,2',6'-tri-N-benzyloxycarbonyl-4-N-acetylfortimicin B (28): [α]_(D)²⁵ + 58.4° (C 1.03, CH₃ OH); IR 1710, 1620, 1500 cm⁻¹ ; NMR (CDCl₃) δ1.16 (C₆ '--CH₃, J=6.0), 2.07 (COCH₃), 2.83 (C₄ --NCH₃), 3.34 (OCH₃),4.81 (H₁ ', J=3.0).

Analysis Calcd. for: C₄₁ H₅₂ N₄ O₁₂ : C, 62.11; H, 6.61; N, 7.07. Found:C, 62.37; H, 6.74; N, 7.00.

EXAMPLE 5 Tetra-N-benzyloxycarbonyl-4-N-glycylglycylfortimicin B (29)

To a stirred suspension of 0.754 g. of1,2'-6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.536 g. ofN-benzyloxycarbonylglycylglycine and 0.622 g. of 1-hydroxybenzotriazolemonohydrate in 4 ml. of tetrahydrofuran was added a solution of 0.418 g.of N,N'-dicyclohexylcarbodiimide in 3 ml. of tetrahydrofuran. Anadditional 3 ml. of tetrahydrofuran was used to rinse all of theN,N'-dicyclohexylcarbodiimide into the reaction vessel. The resultingsuspension was stirred at room temperature for 44 hours. The insolubleN,N'-dicyclohexylurea was then removed by filtration and washedthoroughly with tetrahydrofuran. The filtrate and washings werecombined, and the tetrahydrofuran was evaporated under reduced pressureleaving 1.96 g. of a white glass. The product was chromatographed on acolumn of silica gel. Elution with a solvent system composed ofbenzene-methanol-ethanol-concentrated ammonium hydroxide(23.5:0.7:2.7:0.2 v/v) yielded 0.824 g. oftetra-N-benzyloxycarbonyl-4-N-glycylglycylfortimicin B (20): [α]_(D)²³ + 43° (C 1.0, CH₃ OH); IR 1712, 1638, 1500 cm⁻¹ ; NMR (CDCl₃) δ 1.17(C₆ '--CH₃, J=6), 2.87 (C₄ --NCH₃) 3.32 (OCH₃).

Analysis Calcd. for: C₅₁ H₆₂ N₆ O₁₅ : C, 61.31; H, 6.25; N, 8.41. Found:C, 61.35; H, 6.40; N, 8.28.

EXAMPLE 6 Tetra-N-benzyloxycarbonyl-4-N-sarcosylfortimicin B (30)

To a stirred solution of 2.26 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.855 g. ofN-benzyloxycarbonylsarcosine and 0.982 g. of 1-hydroxybenzotriazolemonohydrate in 12.0 ml. of tetrahydrofuran was added 0.808 g. ofN,N'-dicyclohexylcarbodiimide dissolved in 6.0 ml. of tetrahydrofuran.An additional 6.0 ml. of tetrahydrofuran was used to rinse all theN,N'-dicyclohexylcarbodiimide into the reaction vessel. Stirring wascontinued for 24 hours at room temperature. InsolubleN,N'-dicyclohexylurea was removed by filtration with a sintered glassfunnel. Removal of the tetrahydrofuran under reduced pressure gave ayellow residue which was chromatographed on a column of silica gelprepared and eluted with a solvent system consisting ofbenzene-methanol-95% ethanol-concentrated ammonium hydroxide(23.5:1.4:2.0:0.2 v/v). Fractions enriched intetra-N-benzyloxycarbonyl-4-N-sarcosylfortimicin B (30) were collectedand rechromatographed on a column of Sephadex LH-20 prepared and elutedwith 95% ethanol. Appropriate fractions were combined to give 2.29 g. oftetra-N-benzyloxycarbonyl-4-N-sarcosylfortimicin B (30) as a white foam:[α]_(D) ²⁴ + 49.9° (C 1.0, CH₃ OH); IR 1710, 1635, 1500 cm⁻¹ ; NMR(CDCl₃) δ 1.15 (C₆ ' --CH₃, J=6.8), 2.79 (C₄ --NCH₃), 2.98 (OCH₃), 3.35##STR42## 4.82 (H₁ ', J=3.0).

Analysis Calcd. for: C₅₀ H₆₁ N₅ O₁₄ : C, 62.82; H, 6.43; N, 7.32. Found:C, 62.59; H, 6.47; N, 7.32.

EXAMPLE 7 Tetra-N-benzyloxycarbonyl-4-N-L-phenylalanylglycylfortimicin B(31)

To a stirred solution of 2.00 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 1.284 g. ofN-benzyloxycarbonyl-L-phenylalanylglycine and 0.892 g. of1-hydroxybenzotriazole monohydrate in 10 ml. of tetrahydrofuran wasadded 0.602 g. of N,N'-dicyclohexylcarbodiimide dissolved in 5.0 ml. oftetrahydrofuran. An additional 5.0 ml. of tetrahydrofuran was used torinse all the N,N'-dicyclohexylcarbodiimide into the reaction vessel.Stirring was continued for 20 hours at room temperature. Insolubledicyclohexylurea was removed by filtration through a sintered glassfunnel. The filtrate was concentrated to dryness to leave a yellowresidue. The residue was chromatographed on a column of silica gelprepared and eluted with a solvent system composed ofbenzene-methanol-95% ethanol-concentrated ammonium hydroxide(23.5:1.4:2.0:0.2 v/v). Fractions enriched in the desired component werecollected and evaporated to dryness. The residue was passed through acolumn of Sephadex LH-20 prepared and eluted with 95% ethanol. Fractionscontaining puretetra-N-benzyloxycarbonyl-4-N-L-phenylalanylglycylfortimicin B (31) werecollected and the ethanol was evaporated under reduced pressure to give1.16 g. of product: [α]_(D) ²⁵ + 28.4° (C 1.03, CH₃ OH); IR 1712, 1637,1500 cm⁻¹ ; NMR (CDCl₃) δ 1.16 (C₆ '--CH₃, J=6), 2.80 C₄ --NCH₃), 3.27(OCH₃).

Analysis Calcd. for: C₅₈ H₆₈ N₆ O₁₅ : C, 63.96; H, 6.29; N, 7.72. Found:C, 63.82; H, 6.45; N, 7.71.

EXAMPLE 8 1,2',6'-Tri-N-benzyloxycarbonyl-(N,N-dimethylglycyl)fortimicinB (32)

To a stirred solution of 2.26 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.515 g. ofdimethylglycine and 1.03 g. of 1-hydroxybenzotriazole monohydrate in 6.0ml. of tetrahydrofuran was added 0.840 g. ofN,N'-dicyclohexylcarbodiimide dissolved in 6.0 ml of tetrahydrofuran. Anadditional 6.0 ml. of tetrahydrofuran was used to rinse all theN,N'-dicyclohexylcarbodiimide into the reaction vessel. Triethylamine(1.5 ml.) was added to the reaction mixture and stirring was continuedfor 20 hours at ambient temperature. Insoluble dicyclohexylurea wasremoved by filtration through a sintered glass funnel and the filtratewas taken to dryness. The residue was chromatographed on a column ofsilica gel prepared and eluted with a solvent system composed ofmethylene chloride-95% aqueous methanol-concentrated ammonium hydroxide(18.2:1.8:0.2 v/v). Fractions containing pure1,2',6'-tri-N-benzyloxycarbonyl-(N,N-dimethylglycyl)fortimicin B (32)were collected and evaporated to dryness to give 1.34 g. of a colorlessglass: [α]_(D) ²³ + 46.1° (C 1.0, CH₃ OH); IR 1711, 1630, 1503 cm⁻¹ ;NMR (CDCl₃) δ 1.16 (C₆ '--CH₃, J=6), 2.3 [N(CH₃)₂ ], 2.89 (C₄ --NCH₃),3.06 (COCH₂ --N<), 3.34 (OCH₃), 4.82 (H₁ ', J=3.0).

Analysis Calcd. for: C₄₃ H₅₇ N₅ O₁₂ : C, 61.78; H, 6.87; N, 8.38. Found:C, 61.75; H, 7.02; N, 8.30.

EXAMPLE 9 Tetra-N-benzyloxycarbonyl-4-N-β-alanylfortimicin B (33)

To a stirred solution of 5.52 g. of1,2',6'-tri-N-benzyloxycarbonyl-β-alanine and 1.96 g. of1-hydroxybenzotriazole monohydrate in 24.0 ml. of tetrahydrofuran waswas added 1.62 g. of N,N'-dicyclohexylcarbodiimide dissolved in 12.0 ml.of tetrahydrofuran. An additional 12.0 ml. of tetrahydrofuran was usedto rinse all the N,N'-dicyclohexylcarbodiimide into the reaction vessel.Stirring was continued for 20 hours at room temperature. Insolubledicyclohexylurea was removed by filtration through a sintered glassfunnel. The filtrate was concentrated to dryness under reduced pressureto yield 8.79 g. of a yellow glass. The glass was chromatographed on acolumn of silica gel using a solvent system of benzene-methanol-95%ethanol-concentrated ammonium hydroxide (23.5:1.4:2.0:0.2 v/v).Fractions enriched in the desired product were collected, taken todryness and rechromatographed on a column of Sephadex LH-20 prepared in95% ethanol. Elution with the same solvent gave fractions containing thedesired product. Removal of the ethanol under reduced pressure gave 4.76g. of tetra-N-benzyloxycarbonyl-4-N-β-alanylfortimicin B (33) as a whiteglass: [α]_(D) ²³ + 42.9° (C 0.94, CH₃ OH); IR 1710, 1620, 1503 cm⁻¹ ;NMR (CDCl₃) δ 1.17 (C₆ '--CH₃, J=6), 2.82 (C₄ --NCH₃), 3.28 (OCH₃), 4.78(H₁ ').

Analysis Calcd. for: C₅₀ H₆₁ N₅ O₁₄ : C, 62.82; H, 6.43; N, 7.32. Found:C, 62.11; H, 6.47; N, 7.29.

EXAMPLE 10 Tetra-N-benzyloxycarbonyl-4-N-D-alanylfortimicin B (34)

To a stirred solution of 2.26 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.856 g. ofN-benzyloxycarbonyl-D-alanine and 0.972 g. of 1-hydroxybenzotriazolemonohydrate in 6.0 ml. of tetrahydrofuran, cooled in an ice bath, wasadded 0.816 g. of N,N'-dicyclohexylcarbodiimide dissolved in 6.0 ml. oftetrahydrofuran. An additional 6.0 ml. of tetrahydrofuran was used torinse all the N,N'-dicyclohexylcarbodiimide into the reaction vessel.The reaction was stirred for 1 hour at 0° and then for 18 hours atambient temperature. Insoluble N,N'-dicyclohexylurea was removed byfiltration through a sintered glass funnel and the tetrahydrofuran wasremoved under reduced pressure to give 4.15 g. of a white foam. Theproduct was chromatographed on a column of silica gel prepared andeluted with a solvent system consisting of benzene-methanol-95%ethanol-concentrated ammonium hydroxide (23.5:1.4:2.0:0.2 v/v).Fractions enriched in the desired product were taken to dryness and theresidue repeatedly rechromatographed on a column of silica gel preparedand eluted with a solvent system consisting of cyclohexane-acetone (1:1v/v). Fractions containing puretetra-N-benzyloxycarbonyl-4-N-D-alanylfortimicin B (34) were pooled andthe solvent evaporated to give 0.669 g. of product as a white foam:[α]_(D) ²⁴ + 41.4° (C 1.0, CH₃ OH); IR 1710, 1625, 1498 cm⁻¹ ; NMR(CDCl₃) δ 1.15 (C₆ '--CH₃, J=6.8), ##STR43## 2.88 (C₄ --NCH₃), 3.27(OCH₃), 4.82 (H₁ ', J=3.7).

Analysis Calcd. for: C₅₀ H₆₁ N₅ O₁₄ : C, 62.82; H, 6.43; N, 7.32. Found:C, 62.83; H, 6.59; N, 7.09.

EXAMPLE 11 Tetra-N-benzyloxycarbonyl-4-N-L-alanylfortimicin B (35)

To a stirred solution of 2.26 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.853 g. ofN-benzyloxycarbonyl-L-alanine and 0.963 g. of 1-hydroxybenzotriazolemonohydrate in 6.0 ml. of tetrahydrofuran, cooled in an ice-water bath,was added 0.803 g. of N,N'-dicyclohexylcarbodiimide dissolved in 6.0 ml.of tetrahydrofuran. An additional 6.0 ml. of tetrahydrofuran was used torinse all the N,N'-dicyclohexylcarbodiimide into the reaction vessel.Stirring at 0° was continued for 1 hour and then at ambient temperaturefor 18 hours. Insoluble N,N'-dicyclohexylurea was removed by filtrationand the filtrate concentrated to dryness to give 4.20 g. of a whitefoam. The product was chromatographed on a column of silica gel preparedand eluted with a solvent system consisting of benzene-methanol-95%ethanol-concentrated ammonium hydroxide (23.5:1.4:2.0:0.2 v/v).Fractions containing the major portion of thetetra-N-benzyloxycarbonyl-4-N-L-alanylfortimicin B (35) were collectedand rechromatographed on a column of silica gel prepared and eluted witha solvent system consisting of acetone-hexane (1:1 v/v). Fractionscontaining the desired product were collected and passed through acolumn of Sephadex LH-20 prepared and eluted with 95% ethanol. Fractionscontaining pure tetra-N-benzyloxycarbonyl-4-N-L-alanylfortimicin B (35)were concentrated to dryness to give 1.29 g. of a colorless foam:[α]_(D) ²⁴ + 37.5° (C 1.0, CH₃ OH); IR 1712, 1630, 1500 cm⁻¹ ; NMR(CDCl₃) δ 1.17 (C₆ '--CH₃, J=6.5), ##STR44## 2.97 (C₄ --NCH₃), 3.29(OCH₃), 4.77 (H₁ ', J=3.0).

Analysis Calcd. for: C₅₀ H₆₁ N₅ O₁₄ : C, 62.82; H, 6.43; N, 7.32. Found:C, 62.80; H, 6.58; N, 7.10.

EXAMPLE 12 Tetra-N-benzyloxycarbonyl-4-N-L-alanylglycylfortimicin B (36)

To a stirred solution of 1.09 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3), 0.440 g. ofN-benzyloxycarbonyl-L-alanylglycine and 0.50 g. of1-hydroxybenzotriazole monohydrate in 6.0 ml. of tetrahydrofuran wasadded a solution of 0.416 g. of N,N'-dicyclohexylcarbodiimide in 3.0 ml.of tetrahydrofuran. An additional 3.0 ml. of tetrahydrofuran was used torinse all the N,N'-dicyclohexylcarbodiimide into the reaction vessel.Stirring was continued for 20 hours at ambient temperature. InsolubleN,N'-dicyclohexylurea was removed by filtration through a sintered glassfunnel. The filtrate was concentrated to dryness to give 2.02 of ayellow foam. Pure product was recovered by column chromatography of thereaction mixture on silica gel with a solvent system composed ofbenzene-methanol-95% ethanol-concentrated ammonium hydroxide(23.5:1.4:2.0:0.2 v/v). Fractions containing the desired product wereevaporated to give 1.08 g. oftetra-N-benzyloxycarbonyl-4-N-L-alanylglycylfortimicin B (36): [α]_(D)²⁴ + 30.0° (C 1.02, CH₃ OH); IR 1711, 1640, 1500 cm⁻¹ ; NMR (CDCl₃) δ1.17 (C₆ '--CH₃), ##STR45## 2.85 (C₄ --NCH₃), 3.30 (OCH₃).

Analysis Calcd. for: C₅₂ H₆₄ N₆ O₁₅ : C, 61.35; H, 6.37; N, 8.30. Found:C, 61.68; H, 6.52; N, 8.28.

EXAMPLE 13 Tetra-N-benzyloxycarbonyl-4-N-L-histidylfortimicin B (37)

A solution of 1.50 g. of 1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3)in 5 ml. of ethyl acetate was cooled in an acetone-Dry Ice bath and acold solution of N-benzyloxycarbonyl-L-histidylazide in 19 ml. of ethylacetate, prepared from 1.21 g. ofN-benzyloxycarbonyl-L-histidylhydrazide according to F. Schneider [Z.Physiol. Chem., 320, 82 (1960)] was added with stirring. The reactionmixture was stirred at -15° for 40 minutes, then at 4"C. for 24 hours,and finally at room temperature overnight. Two drops of a concentratedammonium hydroxide solution was evaporated under reduced pressure atroom temperature to leave a residue of 2.36 g. of crude reactionproduct. The latter was chromatographed on 180 g. of silica gel usingmethylene chloride-95 aqueous methanol-concentrated ammonium hydroxide(1170:70:5 v/v) as the eluating solvent. The early chromatographicfractions contained nonpolar substances together with unreacted1,2',6'-tri-N-benzyloxycarbonylfortimicin B (0.35 g.). The residueobtained from the next group of fractions contained a small amount ofstarting material together with the desiredtetra-N-benzyloxycarbonyl-4-N-histidylfortimicin B (37, 1.02 g.). Laterfractions contained 0.30 g. of puretetra-N-benzyloxycarbonyl-4-N-L-histidylfortimicin B (37).

The mixture described above (1.02 g.) containing starting material andthe desired product was rechromatographed on 140 g. of silica gel usingbenzene-methanol-95% ethanol (1174:34:136 v/v) as the eluent.Evaporation of the combined fractions containingtetra-N-benzyloxycarbonyl-4-N-histidylfortimicin B (37) afforded aresidue of 0.75 g. of 37.

A part of the above substance was purified for analysis bychromatography on a Sephadex LH-20 column using 95% ethanol as theeluent. The fractions containing the desired compound were combined,evaporated and the residue was dissolved in chloroform. The chloroformsolution was washed with water. The aqueous layer was separated, theorganic solution was filtered through a sintered glass funnel andevaporated. The residue was pure by TLC: [α]_(D) ²² + 32° (C 1.01,CHCl₃); IR (KBr-pellet): 1710, 1631, 1505 cm⁻¹ ; NMR (CDCl₃)δ 1.15(6'--CH₃); 2.91, 2.93 (C₄ --N--CH₃); 3.22, 3.29 (OCH₃); 5.03, 5.07(Cbz-CH₂); 7.1-7.4 (Cbz-Arom).

Analysis Calcd. for: C₅₃ H₆₃ N₇ O₁₄ : C, 62.28; H, 6.21; N, 9.59. Found:C, 62.05; H, 6.31; N, 9.44.

EXAMPLE 14Tetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-3-aminopropionyl)fortimicinB (38)

The N-hydroxy-5-norbornene-2,3-dicarboximide active ester ofN-benzyloxycarbonyl-DL-2-hydroxy-3-aminopropionic acid was preparedaccording to the general procedure described by M. Fujino, et al. [Chem.Pharm. Bull. Japan, 22, 1857 (1974)]. TheN-Benzyloxycarbonyl-DL-2-hydroxy-3-aminopropionic acid (1.44 g.) wasallowed to react with 1.11 g. ofN-hydroxy-5-norbornene-2,3-dicarboximide in the presence of 1.28 g. ofN,N-dicyclohexylcarbodiimide in 10 ml. of tetrahydrofuran-dioxane (1:1v/v) solution. The N,N'-dicyclohexylurea which was formed in the courseof the above reaction was collected on a filter and the active estersolution was added to a flask containing 2.25 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3). The resulting mixturewas then stirred at room temperature for 2 days. A small amount ofN,N'-dicyclohexylurea was collected on a filter and the filtrate wasevaporated under reduced pressure to afford a residue of 5.46 g. Thesubstance was chromatographed on 270 g. of silica gel withbenzene-methanol-95% ethanol-concentrated ammonium hydroxide(1174:34:136:10 v/v). The early chromatographic fractions contained 1.82g. of the desired product contaminated by a small amount of a less polarimpurity as shown by TLC. The mixture was rechromatographed on 180 g. ofsilica gel using benzene-methanol (85:15 v/v) as the eluent. Evaporationof the appropriate fractions yielded 1.08 g. of the desiredtetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-3-aminopropionyl)fortimicinB (38).

An analytical sample was prepared by chromatography on a Sephadex LH-20column. The product obtained was a mixture of the D- and L- epimers asshown by TLC and NMR: [α]_(D) ²³ + 42° (C 1.07, CH₃ OH); IR (CDCl₃);1705, 1628, 1500 cm⁻¹ ; NMR (CDCl₃)δ 3.03 (C₄ --NCH₃); 3.36, 3.31(OCH₃); 5.0-5.1 Cbz-CH); 7.2-7.4 (Cbz-Arom).

Analysis Calcd. for: C₅₀ H₆₁ N₅ O₁₅ : C, 61.78; H, 6.33; N, 7.20. Found:C, 61.71; H, 6.58; N, 7.27.

The epimers could be separated by chromatography on a Sephadex LH-20column using chloroform-hexane (1:1 v/v) as the eluent. In this manner,thetetra-N-benzyloxycarbonyl-4-N-(C-2-hydroxy-3-aminopropionyl)fortimicin Bas well as thetetra-N-benzyloxycarbonyl-4-N-(L-2-hydroxy-3-aminopropionyl)fortimicin Bcould be obtained in pure form.

EXAMPLE 15 Tetra-N-benzyloxycarbonyl-4-N-L-leucylglycylfortimicin B (39)

A solution of the N-hydroxy-5-norbornene-2,3-dicarboximide active esterof N-benzyloxycarbonyl-L-leucylglycine was prepared according to thegeneral procedure of M. Fujino, et al. [Chem. Pharm. Bull. Japan, 22,1857 (1974)]. A solution of 1.27 g. of N-benzyloxycarbonylleucylglycineand 0.72 g. of N-hydroxy-5-norbornene-2,3-dicarboximide in 5 ml. oftetrahydrofuran was cooled in an ice bath and 0.83 g. ofN,N'-dicyclohexylcarbodiimide was added to the cold solution togetherwith 1 ml. of tetrahydrofuran. The reaction mixture was stirred at lowtemperature for 40 minutes and then at room temperature for 21/2 hours.The N,N'-dicyclohexylurea formed during the reaction was collected on afilter and washed with three 1-ml. portions of tetrahydrofuran.

The solution of the active ester obtained above was allowed to reactwith 1.50 g. of 1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) for 20hours with stirring at room temperature. Evaporation of the solventyielded a residue of 3.59 g. which was chromatographed on 280 g. ofsilica gel using benzene-methanol-95% ethanol-ammonium hydroxide(1174:34:136:10 v/v) as the eluent. A total of 1.76 g. of puretetra-N-benzyloxycarbonyl-4-N-L-leucylglycylfortimicin B (39) wasobtained after evaporation of the solvent from the appropriatefractions.

A part of the product described above was prepared for analysis bychromatography on a Sephadex LH-20: [α]_(D) ²⁷ + 24° (C 1.08, CHCl₃); IR(KBr-pellet) 1710, 1636, 1500 cm⁻¹ ; NMR (CDCl₃) δ 0.92 (Leu-CH₃); 1.17(C₆ '--CH₃, J=6.0), 2.82 (C₄ --NCH₃), 3.30 (OCH₃), 5.0-5.1 (Cbz--CH₂),7.2-7.4 (Cbz-Arom).

Analysis Calcd. for: C₅₅ H₇₀ N₆ O₁₅ : C, 62.60; H, 6.69; N, 7.96. Found:C, 62.31; H, 6.78; N, 7.93.

EXAMPLE 164-N-(N-tert-butyloxycarbonylglycyl)-1,2',6'-tri-N-benzyloxycarbonylfortimicinB (40)

The N-hydroxy-5-norbornene-2,3-dicarboximide active ester ofN-tert-butyloxycarbonylglycine was prepared according to the generalprocedure of M. Fujino et al [Chem. Pharm. Bull. Japan, 22 1857 (1974)].In this case the active ester was isolated and recrystallized from ethylacetate-heptane, m.p. 126°-128°.

A solution prepared from 3.01 g. of1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) and 3.03 g. of the aboveprepared active ester in 10 ml. of chloroform was initially cooled byimmersion in an ice bath. The mixture was then stirred overnight at roomtemperature. Evaporation of the solvent left a residue of 6.84 g. of thecrude coupling product which was purified by chromatography on 270 g. ofsilica gel using benzene-methanol-95% ethanol-concentrated ammoniumhydroxide (1174:34:136:10 v/v) as the eluent. The early chromatographicfractions contained4-N-(N-tert-butyloxycarbonylglycyl)-1,2',6'-tri-N-benzyloxycarbonylfortimicinB (40) contaminated by a small amount of a more polar higher substitutedcompound. Evaporation of the solvent yielded a residue of 3.07 g. of amixture. From the later fractions, 0.49 g. of unreacted1,2',6'-tri-N-benzyloxycarbonylfortimicin B (3) was obtained afterevaporation of the solvent. Repeated rechromatography of the mixture(3.07 g.) containing the desired product on silica gel inbenzene-methanol 85:15 followed by Sephadex LH-20 chromatography using95% ethanol as the eluent afforded 1.07 g. of pure4-N-(N-tert-butyloxycarbonyl)glycyl-1,2',6'-tri-N-benzyloxycarbonylfortimicinB (40): [α]_(D) ²⁶ + 36° (C 1.05, CHCl₃); IR (Kbr disc) 1712, 1640, 1500cm⁻¹ ; NMR (CDCl₃) δ 1.44 (tert-butyloxy-CH₃), 2.82 (C₄ --NCH₃), 3.30(OCH₃); 5.0-5.1 (Cbz-CH₂), 7.2-7.4 (Cbz-Arom).

Analysis Calcd. for: C₄₆ H₆₁ N₅ O₁₄ : C, 60.84; H, 6.77; N, 7.71. Found:C, 60.52; H, 6.99; N, 7.66.

The above mentioned more polar substances contaminating the desiredproduct in the early chromatographic fractions was di-/4-N, 5-0 (or2-0)-tert-butyloxycarbonylglycyl/-1,2',6'-tri-N-benzyloxycarbonylfortimicinB. Purification of this substance provided an analytical sample: [α]_(D)²² + 37° (C 1.01, CHCl₃); IR (KBr-disc) 1710, 1648, 1505 cm⁻¹ ; NMR(CDCl₃) δ 4.9-5.1 (Cbz-CH₂), 7.1-7.4 (Cbz-Arom).

Analysis Calcd. for: C₅₃ H₇₂ N₆ O₁₇ : C, 59.76; H, 6.81; N, 7.89. Found:C, 59.63; H, 7.04; N, 7.86.

EXAMPLE 17 4-N-Glycyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin Btrifluoroacetate salt (41)

A solution of 0.78 g. of4-N-(N-tert-butyloxycarbonylglycyl)-1,2',6'-tri-N-benzyloxycarbonylfortimicinB (40) in 5 ml. of methylene chloride and 5 ml. of trifluoroacetic acidwas stirred at room temperature for 20 minutes. The solution wasevaporated under reduced pressure and the residue was redissolved in 15ml. of methylene chloride and likewise evaporated. The last process wasrepeated six times. The partially deprotected substance was dried overpotassium hydroxide pellets and phosphorous pentoxide under high vacuumfor several hours. The residue of 1.06 g. of4-N-glycyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B trifluoroacetatesalt (41) still contained adhering trifluoroacetic acid in excess ofthat expected for the salt.

EXAMPLE 18Tetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-4-aminobutyryl)glycylfortimicinB (42)

The N-hydroxy-5-norbornene-2,3-dicarboximide active ester ofN-benzyloxycarbonyl DL-2-hydroxy-4-aminoburyic acid was preparedaccording to the procedure of M. Fujino, et al. [Chem. Pharm. Bull.Japan, 22, 1857 (1974)]. To an ice cold solution of 0.40 g. ofN-benzyloxycarbonyl-DL-2-hydroxy-4-aminoburyric acid and 0.32 g. ofN-hydroxy-5-norbornene-2,3-dicarboximide in 3 ml. oftetrahydrofuran-dioxane (1:1 v/v), there was added, with stirring, 0.36g. of N,N'-dicyclohexylcarbodiimide and 1 ml. of the above solventmixture. The solution was stirred in the cold for 30 minutes and then atroom temperature for 2 hours. The N,N'-dicyclohexylurea which formed inthe above reaction was collected on a filter and washed with three 1-ml.portions of tetrahydrofuran-dioxane (1:1 v/v).

The filtrate containing the active ester was collected in a flaskcontaining 4-N-glycyl-1,2',6'-tri-N-benzuloxycarbonylfortimicin Btrifluoroacetate salt (41) and the reaction mixture was immersed into anice-salt bath. Then 0.56 ml. of triethylamine was added to the mixtureto neutralize the trifluoroacetic acid. The reaction mixture was stirredovernight at room temperature. An additional 0.3 ml. of triethylaminewas added and stirring at room temperature was continued for 30 minutes.A small amount of solid was collected on a filter and washed withseveral small portions of tetrahydrofuran-dioxane (1:1 v/v). Evaporationof the filtrate provided a residue of 2.37 g. which was chromatographedon 180 g. of silica gel with benzene-methanol-95% ethanol-concentratedammonium hydroxide (1174:34:136:10 v/v) as the eluent to yield 0.35 g.of product. This substance was rechromatographed on a Sephadex LH-20column in a 95% ethanol solution. Thetetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-4-aminobutyryl)glycylfortimicinB (42) had the following physical constants: [α]_(D) ²⁵ + 29° (C 1.01,CHCl₃); IR (KBr-disc) 1710, 1638, 1510 cm⁻¹ ; NMR (CDCl₃) δ 2.90, 2.99(NCH₃), 3.32 (OCH₃); 5.0-5.1 (Cbz-CH₂); 7.2-7.4 (Cbz-Arom).

Analysis Calcd. for: C₅₃ H₆₆ N₆ O₁₆ : C, 61.02; H, 6.38; N, 8.06. Found:C, 60.80; H, 6.44; N, 8.02.

EXAMPLE 19 Tetra-N-benzyloxycarbonylglycylglycylglycylfortimicin B (43)

The N-hydroxy-5-norbornene-2,3-dicarboximide active ester ofN-benzyloxycarbonylglycylglycine was prepared according to the procedureof M. Fujino, et al. [Chem. Pharm. Bull. Japan, 22, 1857 (1974)]. To anice-cold solution of 0.38 g. of N-benzyloxycarbonylglycylglycine and0.27 g. of N-hydroxy-5-norbornene-2,3-dicarboximide in 4 ml. ofN,N'-dimethylformamide there was added, with stirring, 0.31 g. ofN,N'-dicyclohexylcarbodiimide and 1 ml. of N,N'-dimethylformamide. Themixture was stirred in the cold for 1 hour and at room temperature for 3hours. The N,N'-dicyclohexylurea was collected on a filter and washedwith three 1-ml. portions of N,N'-dimethylformamide.

The filtrate containing the active ester was collected in a flaskcontaining the 4-N-glycyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin Btrifluoroacetate salt (41) freshly prepared from 0.91 g. of4-N-(N-tert-butyloxycarbonylglycyl)-1,2',6'-tri-N-benzyloxycarbonylfortimicinB (40) according to the procedure described above in Example 17. Thereaction mixture was cooled in an ice bath and 0.52 ml. of triethylaminewas added to the cold solution to neutralize the trifluoroacetic acid.The reaction mixture was stirred at room temperature overnight.Evaporation of the solvent yielded a residue of 2.04 g. The substancewas purified by chromatography on 180 g. of silica gel usingbenzene-methanol-95% ethanol-concentrated ammonium hydroxide(1174:34:136:10 v/v) as the eluent. Evaporation of the appropriatechromatographic fractions left a residue of 0.90 g. of the desiredtetra-N-benzyloxycarbonyl-4-N-glycylglycylglycylfortimicin B (43):[α]_(D) ²³ + 44° (C 1.01, CHCl₃); IR (CDCl₃) 1705, 1670, 1505 cm⁻¹ ; NMR(CDCl₃) δ 2.95 (C₄ --NCH₃): 3.33 (OCH₃); 5.0-5.1 (Cbz-CH₂); 7.2-7.4(Cbz-Arom).

Analysis Calcd. for: C₅₃ H₆₅ N₇ O₁₆ : C, 60.27; H, 6.20; N, 9.28. Found:C, 60.09; H, 6.22; N, 9.14.

EXAMPLE 20Tetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-3-aminopropionyl)glycylfortimicinB (44)

To an ice-cold stirred solution of4-N-glycyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B trifluoroacetatesalt, prepared from 0.82 g. of4-N-(N-tert-butyloxycarbonylglycyl)-1,2',6'-tri-N-benzyloxycarbonylfortimicinB according to the procedure described in Example 17, and theN-hydroxy-5-norbornene-2,3-dicarboximide active ester, prepared from0.32 g. of N-benzyloxycarbonyl-DL-2-hydroxy-3-aminopropionic acid asdescribed in Example 14, in 7 ml. of tetrahydrofuran-dioxane (1:1 v/v)there was added 0.4 ml. of triethylamine. The mixture was stirred in thecold for 40 minutes and then overnight at room temperature. The solventwas evaporated to leave a residue of 2.16 g. The residue was purified bychromatography on 180 g. of silica gel using benzene-methanol-95%ethanol-concentrated ammonium hydroxide (1174:34:136:10 v/v) as theeluent. Evaporation of the appropriate fractions led to the isolation of0.83 g. of product. The latter was chromatographed on a Sephadex LH-20column using 95% ethanol as the eluent. A total of 0.74 g. of puretetra-N-benzyloxycarbonyl-4-N-(DL-2-hydroxy-3-aminopropionyl)glycylfortimicin B was obtained (44). An analyticalsample had the following physical constants: [α]_(D) ²³ + 32° (C 1.00,CHCl₃); IR (CDCl₃) 1705, 1636, 1503 cm⁻¹ ; NMR (CDCl₃) δ 2.90, 2.96 (C₄--NCH₃); 3.31 (OCH₃) 5.0-5.1 Cbz-CH₂), 7.2-7.4 (Cbz-Arom).

Analysis Calcd. for: C₅₂ H₆₄ N₆ O₁₆ : C, 60.68; H, 6.27; N, 8.17. Found:C, 60.86; H, 7.47; N, 8.20.

The procedure for removal of the protecting benzyloxycarbonyl groups##STR46## from the per-N-carbobenzyloxycarbonyl derivatives, is asillustrated in Example 21 below, by the conversion oftetra-N-benzyloxycarbonyl-4-N-sarcosylfortimicin B (30) to4-N-sarcosylfortimicin B (13) which is isolated as thetetrahydrochloride salt.

EXAMPLE 21 4-N-Sarcosylfortimicin B (13)

Tetra-N-benzyloxycarbonyl-4-N-sarcosylfortimicin B (30, 0.840 g.)hydrogenolyzed in 150 ml. of 0.2 N hydrochloric acid in methanol (the0.2 N hydrochloric acid solution was prepared by diluting 16.8 ml. ofconcentrated hydrochloric acid to 1000 ml. with methanol) for 4 hoursunder 3 atmospheres of hydrogen in the presence of 0.800 g. of 5%palladium on carbon. The catalyst was removed by filtration and themethanol was evaporated under reduced pressure. Residual water andexcess acid was removed by co-distillation with methanol under reducedpressure to yield 0.512 g. of 4-N-sarcosylfortimicin B (13) as thetetrahydrochloride salt: [α]_(D) ²⁰ + 81.3° (C 1.0, CH₃ OH); IR (KBrdisc) 1640 cm⁻¹ ; NMR (D₂ O) δ 1.84 (C₆ '--CH₃, J=6.6), 3.32 (COCH₂--NCH₃), 3.62 (C₄ --NCH₃), 3.99 (OCH₃), 5.82 (H₁ ', J=3.2).

Mass Spectrum: M⁺. Calcd. for C₁₈ H₃₇ N₅ O₆ : 419.2744. Observed:419.2732

EXAMPLES 22-37

By the procedure of Example 21 above, using the appropriateN-benzyloxycarbonyl protected intermediates (26, 27, 28, 29, 31, 32, 33,34, 35, 36, 39, 42, 37, 43, 44, 38), respectively, described above, thefollowing perhydrochloride salts were prepared:

(9) Fortimicin A tetrahydrochloride,

(10) 4-N-(DL-2-Hydroxy-4-aminobutyryl)fortimicin B tetrahydrochloride,

(11) 4-N-Acetylfortimicin B trihydrochloride,

(12) 4-N-Glycylglycylfortimicin B tetrahydrochloride,

(14) 4-N-L-Phenylalanylglycylfortimicin B tetrahydrochloride,

(15) 4-N-(N,N-Dimethylglycyl)fortimicin B tetrahydrochloride,

(16) 14-N-β-Alanylfortimicin B tetrahydrochloride,

(17) 4-N-D-Alanylfortimicin B tetrahydrochloride,

(18) 4-N-L-Alanylfortimicin B tetrahydrochloride,

(19) 4-N-L-Alanylglycylfortimicin B tetrahydrochloride,

(20) 4-N-L-Leucylglycylfortimicin B tetrahydrochloride,

(21) 4-N-(DL-2-Hydroxy-4-aminobutyrl)glycylfortimicin Btetrahydrochloride,

(22) 4-N-Histidylfortimicin B pentahydrochloride,

(23) 4-N-Glycylglycylglycylfortimicin B tetrahydrochloride,

(24) 4-N-(DL-2-Hydroxy-3-aminopropionyl)glycylfortimicin Btetrahydrochloride, and

(25) 4-N-(DL-2-Hydroxy-3-aminopropionyl) fortimicin Btetrahydrochloride.

The characteristic physical data of these compounds is listed in TableI.

                                      TABLE I                                     __________________________________________________________________________          Rotation   IR            NMR.sup.(b)                                    Compound                                                                            (Methanol) (cm.sup.-1)                                                                       Mass Spectra.sup.(a)                                                                    D.sub.2 O, δ                             __________________________________________________________________________                         M.sup.+.                                                  9    [α].sub.D.sup.23 + 82.3° (C 1.0)                                            1643                                                                              Calcd: 405.2587                                                                         1.79 (C.sub.6 'CH.sub.3), J=7.0),                                   Meas: 405.2617                                                                          3.57 (C.sub.4NCH.sub.3), 3.93                                                 (OCH.sub.3), 5.76 (H.sub.1 ',                                                 J=3.2)                                         10      --       1600                                                                              M.sup.+.H.sub.2 O                                                                       1.80 (C.sub.6 'CH.sub.3, J=6.5)                                     Calcd: 431.2744                                                                         3.32, 3.64 (C.sub.4NCH.sub.3)                                       Meas: 431.2762                                                                          3.94, 4.00 (OCH.sub.3),                                                       5.78, 5.93 (H.sub.1 ', J=3.8,                                                 J=3.6                                                               M.sup.+.                                                 11    [α].sub.D.sup.25 + 87.2 (C 1.04)                                                   1600                                                                              Calcd: 391.2556                                                                         1.80 (C.sub.6 'CH.sub.3, J=6.9),                                    Meas: 391.2553                                                                          2.62 (COCH.sub.3), 3.61                                                       (C.sub.4NCH.sub.3), 3.94 (OCH.sub.3),                                         5.77 (H.sub.1 ', J=3.2)                                             M.sup.+.                                                 12    [α].sub.D.sup.25 + 70.5° (C 1.02)                                           1678                                                                              Calcd: 444.2676                                                                         1.81 (C.sub.6 'CH.sub.3, J=6.4),                                    Meas: 444.2699                                                                          3.62 (C.sub.4NCH.sub.3), 3.95                                                 (OCH.sub.3), 5.79 (H.sub.1 ',                                                 J=3.5)                                                              M.sup.+.                                                 14    [α].sub.D.sup.25 + 76.0° (C 1.06)                                           1674                                                                              Calcd: 553.3350                                                                         1.80 (C.sub.6 'CH.sub.3, J = 6.8),                                  Meas: 553.3329                                                                          3.59 (C.sub.4NCH.sub.3), 3.94                                                 (OCH.sub.3),                                                                  5.77 (H.sub.1 ', J=3.5) 7.85                                                   ##STR47##                                                          M.sup.+.                                                 15    [α].sub.D.sup.25 + 79.3° (C 1.0)                                            1640                                                                              Calcd: 433.2900                                                                         1.81 (C.sub.6 'CH.sub.3 , J=6.4),                                   Meas: 433.2903                                                                          3.44, 3.47 [N(CH.sub.3).sub.2 ],                                              3.56 (C.sub.4NCH.sub.3), 3.95                                                 (OCH.sub.3), 5.80 (H.sub.1 ', J=3.0)                                M.sup.+.                                                 16    [α].sub.D.sup.23 + 61.3° (C 1.0)                                            1610                                                                              Calcd: 419.2744                                                                         1.81 (C.sub.6 'CH.sub.3, J=6.9),                                    Meas: 419.2727                                                                          3.61 (C.sub.4NHC.sub.3) 3.96                                                  (OCH.sub.3), 5.79 (H.sub.1 ',                                                 J=3.0)                                                              M.sup.+.                                                 17    [α].sub.D.sup.20 + 83.2° (C 1.0)                                            1632                                                                              Calcd: 419.2744                                                                         1.81 (C.sub.6 'CH.sub.3, J=7.0),                                    Meas: 419.2723                                                                          2.01 (COCHNH.sub.2 CH.sub.3,                                                  J=6.9), 3.69 (C.sub.4NCH.sub.3),                                              3.94 (OCH.sub.3), 5.77 (H.sub.1 ',                                            J=3.7)                                                              M.sup.+.                                                 18    [α].sub.D.sup.20 + 85.2°  (C 1.02)                                          1640                                                                              Calcd: 419.2744                                                                         1.81 (C.sub.6 'CH.sub.3, J=7.0),                                    Meas: 419.2723                                                                          1.97 (COCHNH.sub.2 CH.sub.3, J=7.0),                                          3.69 (C.sub.4NCH.sub.3), 3.95                                                 (OCH.sub.3),                                                                  5.80 (H.sub.1 ', J=3.8)                                             M.sup.+.                                                 19    [α].sub.D.sup.25 + 76.9° (C 1.0)                                            1674                                                                              Calcd: 476.2958                                                                         1.81 (C.sub.6 'CH.sub.3, J=6.5),                                    Meas: 476.2951                                                                          .04 (COCHNH.sub.2 CH.sub.3),                                                  3.95 (OCH.sub.3), 5.78                                                        (H.sub.1 ', J=3.2)                                                  M.sup.+.                                                 20    [α].sub.D.sup.26 + 62° (C 1.00)                                              1670,                                                                            Calcd: 518.3428                                                                         1.45 (LeuCH.sub.3, J=5.0)                                       1630                                                                              Meas: 518.3454                                                                          1.81 (C.sub.6 'CH.sub.3, J=6.5),                                1487          3.63 (C.sub.4NCH.sub.3), 3.96                                                 (OCH.sub.3),                                                                  5.79 (H.sub.1 ', J=3.5)                                             M.sup.+. 3H.sub.2 O                                      21    [α].sub.D.sup.24 + 58° (C 1.01)                                              1625,                                                                            Calcd: 452.2747                                                                         1.82 (C.sub.6 'CH.sub.3, J=6.5),                                1485                                                                              Meas: 452.2767                                                                          3.65 (C.sub.4NCH.sub.3), 3.97                                                 (OCH.sub.3), 5.80 (H.sub.1 ', J=3.5)                                M.sup.+. H.sub.2 O                                       22    [α].sub.D.sup.25 + 87° (C 0.96)                                              1640,                                                                            Calcd: 467.2856                                                                         1.81 (C.sub.6 'CH.sub.3, J=6.5                                   1590,                                                                            Meas: 467.2869                                                                          3.61 (C.sub.4NCH.sub.3), 3.92                                   1490          (OCH.sub.3), 5.79 (H.sub.1 '                                                  J=3.5), 7.96 (His H-5,                                                        J=1.5), 9.22 (His H-2,                                                        J=1.5)                                                              M.sup.+. OH                                              23    [α].sub.D.sup.25 + 58° (C 1.05)                                              1635,                                                                            Calcd: 502.2989                                                                         1.74 (C.sub.6 'CH.sub.3, J=6.5),                                1485                                                                              Meas: 502.2973                                                                          3.56 (C.sub.4NCH.sub.3), 3.89 (OCH.sub.3)                                     5.81 (H.sub.1 ', J=3.5)                                             M.sup.+.                                                 24    [α].sub.D.sup.26 + 68° (C 1.00)                                              1628,                                                                            Calcd: 492.2907                                                                         1.82 (C.sub.6 'CH.sub.3, J=6.5),                                1485                                                                              Meas: 492.2921                                                                          3.65 (C.sub.4NCH.sub.3), 3.97 (OCH.sub.3)                                     5.78 (H.sub.1 ', J=3.5) -   M.sup.+.                                          H.sub.2 ONH.sub.3                              25    [α].sub.D.sup.27 + 78° (C 1.04)                                              1625,                                                                            Calcd: 400.2322                                                                         1.83 (C.sub.6 'CH.sub.3, J=6.5),                                1487                                                                              Meas: 400.2330                                                                          3.75 (C.sub.4NCH.sub.3), 3.99                                                 (OCH.sub.3), 5.82 (H.sub.1 ',                  __________________________________________________________________________                                   J=3.5)                                          .sup.(a) The mass spectra of the HCl salts of the fortimicin analogs          appear as those of the free bases because of thermal                          dissociation to the free bases prior to volatilizaton in the mass             spectrometer.                                                                 .sup.(b) The 100 MHz NMR-spectra were determined in D.sub.2 O solution        using TMS as an external standard. To convert the                             chemical shifts reported to the Internal TSP-scale; δTMS external =     δTSP internal +0.42 ppm.                                           

EXAMPLES 38-53

In Vitro Antibiotic Activities of 4-N-Acylfortimicin B Derivatives

The in vitro antibiotic activities of the following fortimicin Bderivatives:

(10) 4-N-(DL-2-Hydroxy-4-aminobutyryl)fortimicin B tetrahydrochloride,

(11) 4-N-Acetylfortimicin B trihydrochloride,

(12) 4-N-Glycylglycylfortimicin B tetrahydrochloride,

(13) 4-N-Sarcosylfortimicin B tetrahydrochloride,

(14) 4-N-L-Phenylalanylglycylfortimicin B tetrahydrochloride,

(15) 4-N-(N,N-Dimethylglycyl)fortimicin B tetrahydrochloride,

(16) 4-N-β-Alanylfortimicin B tetrahydrochloride,

(17) 4-N-D-Alanylfortimicin B tetrahydrochloride,

(18) 4-N-L-Alanylfortimicin B tetrahydrochloride,

(19) 4-N-L-Alanylglycylfortimicin B tetrahydrochloride,

(20) 4-N-L-Leucylglycylfortimicin B tetrahydrochloride,

(21) 4-N-(DL-2-Hydroxy-4-aminobutyryl)glycylfortimicin Btetrahydrochloride,

(22) 4-N-L-Histidylfortimicin B pentahydrochloride,

(23) 4-N-glycylglycylglycylfortimicin B tetrahydrochloride,

(24) 4-N-(DL-2-Hydroxy-3-aminopropionyl)glycylfortimicin Btetrahydrochloride, and

(25) 4-N-(DL-2-Hydroxy-3-aminopropionyl)fortimicin B tetrahydrochloride

are listed in Table II, below.

The in vitro antibiotic activities were determined by a two-fold agardilution method using Mueller-Hinton agar, 10 ml. per Petri dish. Theagar was inoculated with one loopful (0.001 ml. loop) of a 1:10 dilutionof a 24 hour broth culture of the indicated test organism and incubatedat 37° C. for 24 hours. Fortimicin A disulfate salt was used as thecontrol antibiotic. The activities are listed in Table II. Minimuminhibitory concentrations (MIC) are expressed in mcg./ml.

                                      TABLE II                                    __________________________________________________________________________    In Vitro Antibiotic Activity of 4-N-Acylfortimicin B Derivatives                                       Compounds                                            Organism         Fortimicin A                                                                          (10)   (11)                                          __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    100    >100                                          Streptococcus faecalis 10541                                                                   50      >100   >100                                          Enterobacter aerogenes 13048                                                                   1.56    >100   >100                                          Escherichia coli Juhl                                                                          3.1     >100   >100                                          Escherichia coli BL3676 (Resist)                                                               12.5    >100   >100                                          Klebsiella pneumoniae 10031                                                                    1.56    >100   >100                                          Klebsiella pneumonlae KY4262                                                                   6.2     >100   >100                                          Providencia 1577 1.56    >100   >100                                          Pseudomonas aeruginosa BMH #10                                                                 0.78    50     >100                                          Pseudomonas aeruginosa KY8512                                                                  6.2     >100   >100                                          Pseudomonas aeruginosa KY8516                                                                  25      >100   >100                                          Pseudomonas aeruginosa 209                                                                     >100    >100   >100                                          Salmonella typhimurium Ed. #9                                                                  1.56    100    >100                                          Serratia marcescens 4003                                                                       1.56    100    >100                                          Shigella sonnei 9290                                                                           6.2     >100   >100                                          Proteus rettgeri U 6333                                                                        25      >100   >100                                          Proteus vulgaris Abbott JJ                                                                     3.1     >100   >100                                          Proteus mirabilis Fin. #9                                                                      6.2     >100   >100                                                                   Compounds                                            Organism         Fortimicin A                                                                          (12)   (13)                                          __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    12.5   3.1                                           Streptococcus faecalis 10541                                                                   50      >100   100                                           Enterobacter aerogenes 13048                                                                   1.56    25     6.2                                           Escherichia coli Juhl                                                                          3.1     25     6.2                                           Escherichia coli BL3676 (Resist)                                                               12.5    50     25                                            Klebsiella pneumoniae 10031                                                                    1.56    25     6.2                                           Klebsiella pneumoniae KY 4262                                                                  6.2     50     25                                            Providencia 1577 1.56    50     25                                            Pseudomonas aeruginosa BMH #10                                                                 0.39    1.56   0.78                                          Pseudomonas aeruginosa KY8512                                                                  6.2     50     25                                            Pseudomonas aeruginosa KY8516                                                                  25      >100   50                                            Pseudomonas aeruginosa 209                                                                     >100    >100   >100                                          Salmonella typhimurium Ed. #9                                                                  1.56    3.1    3.1                                           Serratia marcescens 4003                                                                       1.56    6.2    3.1                                           Shigella sonnei 9290                                                                           6.2     25     6.2                                           Proteus rettgeri U 6333                                                                        25      100    100                                           Proteus vulgaris Abbott JJ                                                                     3.1     25     6.2                                           Proteus mirabilis Fin. #9                                                                      6.2     50     12.5                                                                   Compound                                             Organism         Fortimicin A                                                                          (14)                                                 __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    >100                                                 Streptococcus faecalis 10541                                                                   50      >100                                                 Enterobacter aerogenes 13048                                                                   1.56    >100                                                 Escherichia coli Juhl                                                                          3.1     100                                                  Escherichia coli BL3676 (Resist)                                                               12.5    >100                                                 Klebsiella pneumoniae 10031                                                                    1.56    >100                                                 Klebsiella pneumoniae KY 4262                                                                  6.2     >100                                                 Providencia 1577 1.56    >100                                                 Pseudomonas aeruginosa BMH #10                                                                 0.78    3.1                                                  Pseudomonas aeruginosa KY8512                                                                  6.2     >100                                                 Pseudomonas aeruginosa KY8516                                                                  25      >100                                                 Pseudomonas aeruginosa 209                                                                     >100    >100                                                 Salmonella typhimurium Ed. #9                                                                  1.56    >100                                                 Serratia marcescens 4003                                                                       1.56    100                                                  Shigella sonnei 9290                                                                           6.2     50                                                   Proteus rettgeri U6333                                                                         25      >100                                                 Proteus vulgaris Abbott JJ                                                                     3.1     >100                                                 Proteus mirabilis Fin. #9                                                                      6.2     >100                                                                          Compounds                                            Organism         Fortimicin A                                                                          (15)   (16)                                          __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    12.5   3.1                                           Streptococcus faecalis 10541                                                                   50      >100   100                                           Enterobacter aerogenes 13048                                                                   1.56    >100   6.2                                           Escherichia coli Juhl                                                                          3.1     25     6.2                                           Escherichia coli BL3676 (Resist)                                                               12.5    >100   25                                            Klebsiella pneumoniae 10031                                                                    1.56    >100   6.2                                           Klebsiella pneumoniae KY4262                                                                   6.2     >100   25                                            Providencia 1577 1.56    >100   25                                            Pseudomonas aeruginosa BMH #10                                                                 0.78    6.2    0.78                                          Pseudomonas aeruginosa KY8512                                                                  6.2     >100   25                                            Pseudomonas aeruginosa KY8516                                                                  25      >100   50                                            Pseudomonas aeruginosa 209                                                                     >100    100    >100                                          Salmonella typhimurium Ed. #9                                                                  1.56    25     1.56                                          Serratia marcescens 4003                                                                       1.56    25     1.56                                          Shigella sonnei 9290                                                                           6.2     50     6.2                                           Proteus rettgeri U 6333                                                                        25      >100   100                                           Proteus vulgaris Abbott JJ                                                                     3.1     50     12.5                                          Proteus mirabilis Fin. #9                                                                      6.2     >100   12.5                                                                   Compounds                                            Organism         Fortimicin A                                                                          (17)   (18)                                          __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    12.5   12.5                                          Streptococcus faecalis 10541                                                                   50      >100   >100                                          Enterobacter aerogenes 13048                                                                   1.56    100    25                                            Escherichia coli Juhl                                                                          3.1     100    25                                            Escherichia coli BL3676 (Resist)                                                               12.5    >100   50                                            Klebsiella pneumoniae 10031                                                                    1.56    >100   50                                            Klebsiella pneumonia KY4262                                                                    6.2     >100   >100                                          Providencia 1577 1.56    >100   50                                            Pseudomonas aeruginosa BMH #10                                                                 0.78    100    1.56                                          Pseudomonas aeruginosa KY8512                                                                  6.2     100    50                                            Pseudomonas aeruginosa KY8516                                                                  25      > 100  >100                                          Pseudomonas aeruginosa 209                                                                     >100    >100   >100                                          Salmonella typhimurium Ed. #9                                                                  1.56    50     25                                            Serratia marcescens 4003                                                                       1.56    25     6.2                                           Shigella sonnei 9290                                                                           6.2     50     12.5                                          Proteus rettgeri U 6333                                                                        25      >100   >100                                          Proteus vulgaris Abbot JJ                                                                      3.1     100    12.5                                          Proteus mirabilis Fin. #9                                                                      6.2     50     25                                                                     Compound                                             Organism         Fortimicin A                                                                          (19)                                                 __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    50                                                   Streptococcus faecalis 10541                                                                   50      >100                                                 Enterobacter aerogenes 13048                                                                   1.56    12.5                                                 Escherichia coli Juhl                                                                          3.1     25                                                   Escherichia coli BL3676 (Resist)                                                               12.5    >100                                                 Klebsiella pneumoniae 10031                                                                    1.56    50                                                   Klebsiella pneumoniae KY4262                                                                   6.2     >100                                                 Providencia 1577 1.56    12.5                                                 Pseudomonas aeruginosa BMH #10                                                                 0.78    1.56                                                 Pseudomonas aeruginosa KY8512                                                                  6.2     50                                                   Pseudomonas aeruginosa KY8516                                                                  25      100                                                  Pseudomonas aeruginosa 209                                                                     >100    >100                                                 Salmonella typhimurium Ed. #9                                                                  1.56    6.2                                                  Serratia marcescens 4003                                                                       1.56    6.2                                                  Shigella sonnei 9290                                                                           6.2     12.5                                                 Proteus rettgeri U 6333                                                                        25      50                                                   Proteus vulgaris Abbott JJ                                                                     3.1     12.5                                                 Proteus mirabilis Fin. #9                                                                      6.2     25                                                                            Compounds                                            Organism         Fortimicin A                                                                          (20)   (21)                                          __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    6.2    6.2                                           Streptococcus faecalis 10541                                                                   50      >100   >100                                          Enterobacter aerogenes 13048                                                                   3.1     50     25                                            Escherichia coli Juhl                                                                          3.1     12.5   25                                            Escherichia coli BL3676 (Resist)                                                               25      25     100                                           Klebsiella pneumoniae 10031                                                                    1.56    50     25                                            Klebsiella pneumoniae KY4262                                                                   6.2     50     100                                           Providencia 1577 12.5    >100   100                                           Pseudomonas aeruginosa BMH #10                                                                 0.39    1.56   6.2                                           Pseudomonas aeruginosa KY8512                                                                  6.2     100    100                                           Pseudomonas aeruginosa KY8516                                                                  12.5    >100   >100                                          Pseudomonas aeruginosa 209                                                                     >100    >100   >100                                          Salmonella typhimurium Ed. #9                                                                  1.56    12.5   12.5                                          Serratia marcescens 4003                                                                       0.78    12.5   12.5                                          Shigella sonnei 9290                                                                           3.1     25     50                                            Proteus rettgeri U 6333                                                                        25      100    >100                                          Proteus vulgaris Abbott JJ                                                                     6.2     50     50                                            Proteus mirabilis Fin. #9                                                                      6.2     >100   >100                                                                   Compound                                             Organism         Fortimicin A                                                                          (22)                                                 __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    50                                                   Streptococcus faecalis 10541                                                                   50      >100                                                 Enterobacter aerogenes  13048                                                                  3.1     >100                                                 Escherichia coli Juhl                                                                          3.1     >100                                                 Escherichia coli BL3676 (Resist)                                                               25      >100                                                 Klebsiella pneumoniae 10031                                                                    1.56    >100                                                 Klebsiella pneumoniae KY4262                                                                   6.2     >100                                                 Providencia 1577 12.5    >100                                                 Pseudomonas aeruginosa BMH #10                                                                 0.39    50                                                   Pseudomonas aeruginosa KY8512                                                                  6.2     >100                                                 Pseudomonas aeruginosa KY8516                                                                  12.5    >100                                                 Pseudomonas aeruginosa 209                                                                     >100    >100                                                 Salmonella typhimurium Ed. #9                                                                  1.56    >100                                                 Serratia marcescens 4003                                                                       0.78    >100                                                 Shigella sonnei 9290                                                                           3.1     >100                                                 Proteus rettgeri U 6333                                                                        25      >100                                                 Proteus vulgaris Abbott JJ                                                                     6.2     >100                                                 Proteus mirabilis Fin. #9                                                                      6.2     >100                                                                          Compounds                                             Organism        Fortimicin A                                                                          (23)   (24)                                          __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    6.2    3.1                                           Streptococcus faecalis 10541                                                                   50      >100   >100                                          Enterobacter aerogenes 13048                                                                   3.1     25     25                                            Escherichia coli Juhl                                                                          3.1     50     25                                            Escherichia coli BL3676 (Resist)                                                               25      100    100                                           Klebsiella pneumoniae 10031                                                                    1.56    50     25                                            Klebsiella pneumoniae  KY4262                                                                  6.2     >100   100                                           Providencia 1577 1.56    25     25                                            Pseudomonas aeruginosa BMH #10                                                                 0.78    3.1    3.1                                           Pseudomonas aeruginosa KY8512                                                                  12.5    100    >100                                          Pseudomonas aeruginosa KY8516                                                                  50      >100   >100                                          Pseudomonas aeruginosa 209                                                                     >100    >100   >100                                          Samonella typhimurium Ed. #9                                                                   3.1     12.5   12.5                                          Serratia marcescens 4003                                                                       1.56    25     12.5                                          Shigella sonnei 9290                                                                           6.2     100    50                                            Proteus rettgeri U 6333                                                                        25      >100   >100                                          Proteus vulgaris Abbott JJ                                                                     3.1     50     25                                            Proteus mirabilis Fin. #9                                                                      6.2     >100   50                                                                     Compound                                             Organism         Fortimicin A                                                                          (25)                                                 __________________________________________________________________________    Staphylococcus aureus Smith                                                                    0.78    25                                                   Streptococcus faecalis 10541                                                                   50      >100                                                 Enterobacter aerogenes 13048                                                                   3.1     50                                                   Escherichia coli Juhl                                                                          3.1     50                                                   Escherichia coli BL3676 (Resist)                                                               25      >100                                                 Klebsiella pneumoniae 10031                                                                    1.56    50                                                   Klebsiella pneumoniae KY4262                                                                   6.2     >100                                                 Providencia 1577 1.56    100                                                  Pseudomonas aeruginosa BMH #10                                                                 0.78    6.2                                                  Pseudomonas aeruginosa KY8512                                                                  12.5    >100                                                 Pseudomonas aeruginosa KY8516                                                                  50      >100                                                 Pseudomonas aeruginosa 209                                                                     >100    >100                                                 Salmonella typhimurium Ed. #9                                                                  3.1     25                                                   Serratia marcescens 4003                                                                       1.56    25                                                   Shigella sonnei 9290                                                                           6.2     50                                                   Proteus rettgeri U 6333                                                                        25      >100                                                 Proteus vulgaris Abbott JJ                                                                     3.1     50                                                   Proteus mirabilis Fin. #9                                                                      6.2     50                                                   __________________________________________________________________________

The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, at least one of thecompounds of this invention which exhibit antimicrobial activity inassociation with the pharmaceutical carrier or diluent. The compounds ofthis invention can be administered by oral or parenteral routes ofadministration, i.e., intramuscular, intravenous, or subcutaneous routesof administration, or rectal administration, and can be formulated indosage forms suitable for each route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert diluent such as sucrose,lactose or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water.Besides inert diluents, such compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying anddispersing agents. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, by irradiating the compositions, or by heatingthe compositions. They can also be manufactured in the form of sterilewater, solid compositions which can be dissolved in sterile water, orsome other sterile injectable medium immediately before use.

Compositions for rectal administration are preferably suppositorieswhich may contain, in addition to the active substance, excipients suchas cocoa butter or a suppository wax.

The dosage of active ingredients in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredients be such that suitable dosage form is obtained. The selecteddosage depends upon the desired therapeutical effect, the route ofadministration and the duration of treatment desired.

We claim:
 1. A method of preparing a 4-N-acylfortimicin B derivative ofthe structure ##STR48## wherein R is acyl, aminoacyl,N-monoloweralkylaminoacyl, N,N-diloweralkylaminoacyl,hydroxy-substituted aminoacyl or substituted aminoacyl of the formula##STR49## where R¹ is an acyl radical derived from an amino acid or ashort peptide, and the pharmaceutically acceptable salts thereof, whichmethod comprises the steps of:(a) preparing1,2',6'-tri-N-benzyloxycarbonylfortimicin B by acylating fortimicin Bwith N-(benzyloxycarbonyloxy)succinimide, benzyloxycarbonyl chloride orO-(benzyloxycarbonyl)p-nitrophenol in a solvent selected from the groupconsisting of methanol, methanol-water, ethanol, ethanol-water,N,N-dimethylformamide, N-N-dimethyl-formamide-water,tetrahydrofuran-water and dioxane-water at a temperature between -10°and 25° C. for 12 to 48 hours; (b) acylating said1,2',6'-tri-N-benzyloxycarbonylfortimicin B with a reagent of theformula

    R.sup.4 --Y

wherein R⁴ is an acyl, N,N-diloweralkylaminoacyl or or an acyl groupderived from an N-benzyloxycarbonyl protected amino acid or a shortpeptide, and Y is an activating group selected from the group consistingof ##STR50## in a solvent selected from the group consisting oftetrahydrofuran, dioxane, chloroform or N,N-dimethylformamide at atemperature between -10° and 25° C. in the presence of triethylamine ortri-n-butylamine for 15 to 48 hours to provide4-N-acyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B of the formula##STR51## wherein R⁴ is as defined above; and (c) hydrogenolyzing saidbenzyloxycarbonyl groups of said4-N-acyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B in 0.1 to 0.6 Nmethanolic hydrochloric acid in the presence of a 2 to 10% palladium oncarbon catalyst under 1 to 10 atmospheres of hydrogen gas at atemperature between 10° and 25° C. to form the desired4-N-acylfortimicin B derivative.
 2. The method of claim 1, wherein said1,2',6'-tri-N-benzyloxycarbonylfortimicin B is prepared by reactingfortimicin B with N-(benzyloxycarbonyloxy) succinimide in amethanol-water solution at a temperature of 0° C. for 3 hours, and thenat room temperature for 22 hours.